NOTE: SeMet is TOXIC. The culture supernatant should be collected for disposal as hazardous waste. Fill the waste bottles to shoulder-height, not all the way to the screw cap. Use the Online Tag Program to generate hazardous waste tags as soon as you start generating the hazardous waste and affix the labels to the waste bottles immediately.

Refer to your lab-specific Standard Operating Procedure (SOP) for the proper way to work with SeMet. At a minimum you will need to use the appropriate Personal Protective Equipment (PPE) which includes the use of nitrile gloves, lab coat, and full-coverage shoes.

Stock solutions

1. Thiamine, 1%0.5 g/50 ml (0.22um filtered)

2. Antibiotic(0.22um filtered)

3. MgsO4, 1M13.32 g/50ml (0.22um filtered)

4. Zn-EDTA:

I ) EDTA250 mg/50ml

II) Zn(Ac)2420 mg/50ml

First dissolve separately and then combine (0.22um filtered)

5. Trace Elements solution

H3BO32.5 g/l

CoCl2*6H2O2 g/l

CuCl2*2H2O1.13 g/l

MnCl2.”H2O9.8 g/l

Na2MoO4*2H2O2 g/l

To dissolve, lower the pH with the citric acid or HCl (0.22um filtered)

6.Glucose, 20%100 g/ 500ml (autoclave separately)

For 1 litre of medium add:

400mgAla, Gln, Glu, Arg, Gly, Ile, Val

255mgAsp, Met

125mgCytosine, Guanosine, Uracil

100mg Asn, Leu, His, Lys, Pro, Thr, Try

50mgPhe, Thymine, Thymidine

10mgCaCl2

2g NaAc

10gK2HPO4

1gCitric Acid

1.3ml Trace element solution

36mgFerrous citrate (dissolve in 120ul of conc. HCl)

1mlZn-EDTA solution

1.6gSer

1gNH4Cl

Adjust the pH to 7.0

Autoclave

To make the final ready media, add to the above autoclaved media :

1. Glucose25 ml

2. Thiamin560 ul

3. Antibiotic Half of the usual amount

4. MgSO42 ml

5. 50mgCys, Trp, Nicotinic acid

6. 0.1mg Biotin

Make over-day culture from freshly transformed cells in 5 ml of LB with antibiotic.

Inoculate 100ul of LB to the 100ml of Minimal Media with antibiotic.

Grow overnight.

Add 1 litre of Minimal Media to the 100ml overnight culture with antibiotic.

Induce at of 0.7- 0.8 with the IPTG (final conc. 1mM).

Grow the culture for 5 – 7 hrs. and spin it.

# Add the desired amino acid at the time of induction as well (same amount as added before, 0.22um filtered).